RESUMO
BACKGROUND: Islet amyloid polypeptide (IAPP) or amylin deposits can be found in the islets of type 2 diabetes patients. The peptide is suggested to be involved in the etiology of the disease through formation of amyloid deposits and destruction of ß islet cells, though the underlying molecular events leading from IAPP deposition to ß cell death are still largely unknown. RESULTS: We used OFFGEL™ proteomics to study how IAPP exposure affects the proteome of rat pancreatic insulinoma Rin-5F cells. The OFFGEL™ methodology is highly effective at generating quantitative data on hundreds of proteins affected by IAPP, with its accuracy confirmed by In Cell Western and Quantitative Real Time PCR results. Combining data on individual proteins identifies pathways and protein complexes affected by IAPP. IAPP disrupts protein synthesis and degradation, and induces oxidative stress. It causes decreases in protein transport and localization. IAPP disrupts the regulation of ubiquitin-dependent protein degradation and increases catabolic processes. IAPP causes decreases in protein transport and localization, and affects the cytoskeleton, DNA repair and oxidative stress. CONCLUSIONS: Results are consistent with a model where IAPP aggregates overwhelm the ability of a cell to degrade proteins via the ubiquitin system. Ultimately this leads to apoptosis. IAPP aggregates may be also toxic to the cell by causing oxidative stress, leading to DNA damage or by decreasing protein transport. The reversal of any of these effects, perhaps by targeting proteins which alter in response to IAPP, may be beneficial for type II diabetes.
Assuntos
Polipeptídeo Amiloide das Ilhotas Pancreáticas/farmacologia , Proteoma/efeitos dos fármacos , Animais , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Cromatografia Líquida de Alta Pressão , Reparo do DNA/efeitos dos fármacos , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Espectrometria de Massas , Estresse Oxidativo/efeitos dos fármacos , Proteoma/genética , Proteoma/metabolismo , RatosRESUMO
Defining intracellular protein concentration is critical in molecular systems biology. Although strategies for determining relative protein changes are available, defining robust absolute values in copies per cell has proven significantly more challenging. Here we present a reference data set quantifying over 1800Saccharomyces cerevisiaeproteins by direct means using protein-specific stable-isotope labeled internal standards and selected reaction monitoring (SRM) mass spectrometry, far exceeding any previous study. This was achieved by careful design of over 100 QconCAT recombinant proteins as standards, defining 1167 proteins in terms of copies per cell and upper limits on a further 668, with robust CVs routinely less than 20%. The selected reaction monitoring-derived proteome is compared with existing quantitative data sets, highlighting the disparities between methodologies. Coupled with a quantification of the transcriptome by RNA-seq taken from the same cells, these data support revised estimates of several fundamental molecular parameters: a total protein count of â¼100 million molecules-per-cell, a median of â¼1000 proteins-per-transcript, and a linear model of protein translation explaining 70% of the variance in translation rate. This work contributes a "gold-standard" reference yeast proteome (including 532 values based on high quality, dual peptide quantification) that can be widely used in systems models and for other comparative studies.
Assuntos
Espectrometria de Massas/métodos , Proteômica/métodos , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Perfilação da Expressão Gênica/métodos , Marcação por Isótopo , Modelos Lineares , Espectrometria de Massas/normas , Proteômica/normas , Proteínas Recombinantes/metabolismo , Reprodutibilidade dos Testes , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Análise de Sequência de RNA/métodosRESUMO
The mechanisms by which RNA-binding proteins control the translation of subsets of mRNAs are not yet clear. Slf1p and Sro9p are atypical-La motif containing proteins which are members of a superfamily of RNA-binding proteins conserved in eukaryotes. RIP-Seq analysis of these two yeast proteins identified overlapping and distinct sets of mRNA targets, including highly translated mRNAs such as those encoding ribosomal proteins. In paralell, transcriptome analysis of slf1Δ and sro9Δ mutant strains indicated altered gene expression in similar functional classes of mRNAs following loss of each factor. The loss of SLF1 had a greater impact on the transcriptome, and in particular, revealed changes in genes involved in the oxidative stress response. slf1Δ cells are more sensitive to oxidants and RIP-Seq analysis of oxidatively stressed cells enriched Slf1p targets encoding antioxidants and other proteins required for oxidant tolerance. To quantify these effects at the protein level, we used label-free mass spectrometry to compare the proteomes of wild-type and slf1Δ strains following oxidative stress. This analysis identified several proteins which are normally induced in response to hydrogen peroxide, but where this increase is attenuated in the slf1Δ mutant. Importantly, a significant number of the mRNAs encoding these targets were also identified as Slf1p-mRNA targets. We show that Slf1p remains associated with the few translating ribosomes following hydrogen peroxide stress and that Slf1p co-immunoprecipitates ribosomes and members of the eIF4E/eIF4G/Pab1p 'closed loop' complex suggesting that Slf1p interacts with actively translated mRNAs following stress. Finally, mutational analysis of SLF1 revealed a novel ribosome interacting domain in Slf1p, independent of its RNA binding La-motif. Together, our results indicate that Slf1p mediates a translational response to oxidative stress via mRNA-specific translational control.
Assuntos
Biossíntese de Proteínas/genética , Proteínas de Ligação a RNA/genética , Ribossomos/genética , Proteínas de Saccharomyces cerevisiae/genética , Regulação Fúngica da Expressão Gênica , Peróxido de Hidrogênio/metabolismo , Estresse Oxidativo/genética , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , Proteínas de Ligação a RNA/biossíntese , Saccharomyces cerevisiae , Proteínas de Saccharomyces cerevisiae/biossíntese , Análise de Sequência de RNARESUMO
Translation initiation factor eIF4E mediates mRNA selection for protein synthesis via the mRNA 5'cap. A family of binding proteins, termed the 4E-BPs, interact with eIF4E to hinder ribosome recruitment. Mechanisms underlying mRNA specificity for 4E-BP control remain poorly understood. Saccharomyces cerevisiae 4E-BPs, Caf20p and Eap1p, each regulate an overlapping set of mRNAs. We undertook global approaches to identify protein and RNA partners of both 4E-BPs by immunoprecipitation of tagged proteins combined with mass spectrometry or next-generation sequencing. Unexpectedly, mass spectrometry indicated that the 4E-BPs associate with many ribosomal proteins. 80S ribosome and polysome association was independently confirmed and was not dependent upon interaction with eIF4E, as mutated forms of both Caf20p and Eap1p with disrupted eIF4E-binding motifs retain ribosome interaction. Whole-cell proteomics revealed Caf20p mutations cause both up and down-regulation of proteins and that many changes were independent of the 4E-binding motif. Investigations into Caf20p mRNA targets by immunoprecipitation followed by RNA sequencing revealed a strong association between Caf20p and mRNAs involved in transcription and cell cycle processes, consistent with observed cell cycle phenotypes of mutant strains. A core set of over 500 Caf20p-interacting mRNAs comprised of both eIF4E-dependent (75%) and eIF4E-independent targets (25%), which differ in sequence attributes. eIF4E-independent mRNAs share a 3' UTR motif. Caf20p binds all tested motif-containing 3' UTRs. Caf20p and the 3'UTR combine to influence ERS1 mRNA polysome association consistent with Caf20p contributing to translational control. Finally ERS1 3'UTR confers Caf20-dependent repression of expression to a heterologous reporter gene. Taken together, these data reveal conserved features of eIF4E-dependent Caf20p mRNA targets and uncover a novel eIF4E-independent mode of Caf20p binding to mRNAs that extends the regulatory role of Caf20p in the mRNA-specific repression of protein synthesis beyond its interaction with eIF4E.
Assuntos
Repressão Epigenética , Fator de Iniciação 4E em Eucariotos/metabolismo , Regulação Fúngica da Expressão Gênica , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/genética , Fatores de Transcrição/metabolismo , Regulação para Baixo , Fator de Iniciação 4E em Eucariotos/genética , Fator de Iniciação Eucariótico 4G/genética , Fator de Iniciação Eucariótico 4G/metabolismo , Imunoprecipitação , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Fases de Leitura Aberta , Ligação Proteica , Biossíntese de Proteínas , Proteômica , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Proteínas de Ligação a RNA , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Análise de Sequência de RNA , Espectrometria de Massas em Tandem , Fatores de Transcrição/genéticaRESUMO
The relocalization of translationally repressed mRNAs to mRNA processing bodies Pbodies is a key consequence of cellular stress across many systems. Pbodies harbor mRNA degradation components and are implicated in mRNA decay, but the relative timing and control of mRNA relocalization to Pbodies is poorly understood. We used the MS2GFP system to follow the movement of specific endogenous mRNAs in live Saccharomyces cerevisiae cells after nutritional stress. It appears that the relocalization of mRNA to Pbodies after stress is biphasic some mRNAs are present early, whereas others are recruited much later concomitant with recruitment of translation initiation factors, such as eIF4E. We also find that Bfr1p is a latephaselocalizing Pbody protein that is important for the delayed entry of certain mRNAS to Pbodies. Therefore, for the mRNAs tested, relocalization to Pbodies varies both in terms of the kinetics and factor requirements. This work highlights a potential new regulatory juncture in gene expression that would facilitate the overall rationalization of protein content required for adaptation to stress.
Assuntos
RNA Fúngico/metabolismo , RNA Mensageiro/metabolismo , Proteínas Repressoras/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Regulação Fúngica da Expressão Gênica , Estabilidade de RNA , Transporte de RNA , Saccharomyces cerevisiae/genética , Estresse FisiológicoRESUMO
In response to stress, the translation of many mRNAs in yeast can change in a fashion discordant with the general repression of translation. Here, we use machine learning to mine the properties of these mRNAs to determine specific translation control signals. We find a strong association between transcripts acutely translationally repressed under oxidative stress and those associated with the RNA-binding protein Puf3p, a known regulator of cellular mRNAs encoding proteins targeted to mitochondria. Under oxidative stress, a PUF3 deleted strain exhibits more robust growth than wild-type cells and the shift in translation from polysomes to monosomes is attenuated, suggesting puf3Δ cells perceive less stress. In agreement, the ratio of reduced:oxidized glutathione, a major antioxidant and indicator of cellular redox state, is increased in unstressed puf3Δ cells but remains lower under stress. In untreated conditions, Puf3p migrates with polysomes rather than ribosome-free fractions, but this is lost under stress. Finally, reverse transcriptase-polymerase chain reaction (RT-PCR) of Puf3p targets following affinity purification shows Puf3p-mRNA associations are maintained or increased under oxidative stress. Collectively, these results point to Puf3p acting as a translational repressor in a manner exceeding the global translational response, possibly by temporarily limiting synthesis of new mitochondrial proteins as cells adapt to the stress.
Assuntos
Regulação Fúngica da Expressão Gênica , Estresse Oxidativo/genética , Biossíntese de Proteínas , Proteínas de Ligação a RNA/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Deleção de Genes , Glutationa/metabolismo , Oxirredução , Polirribossomos/metabolismo , RNA Mensageiro/metabolismo , Proteínas de Ligação a RNA/genética , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genéticaRESUMO
The crystal structure of AdhP, a recombinantly expressed alcohol dehydrogenase from Escherichia coli K-12 (substrain MG1655), was determined to 2.01 Å resolution. The structure, which was solved using molecular replacement, also included the structural and catalytic zinc ions and the cofactor nicotinamide adenine dinucleotide (NAD). The crystals belonged to space group P21, with unit-cell parameters a = 68.18, b = 118.92, c = 97.87â Å, ß = 106.41°. The final R factor and Rfree were 0.138 and 0.184, respectively. The structure of the active site of AdhP suggested a number of residues that may participate in a proton relay, and the overall structure of AdhP, including the coordination to structural and active-site zinc ions, is similar to those of other tetrameric alcohol dehydrogenase enzymes.
Assuntos
Álcool Desidrogenase/química , Álcool Desidrogenase/metabolismo , Escherichia coli/enzimologia , NAD/metabolismo , Álcool Desidrogenase/genética , Sequência de Aminoácidos , Sítios de Ligação , Catálise , Domínio Catalítico , Cristalização , Cristalografia por Raios X , Etanol/farmacologia , Modelos Moleculares , Dados de Sequência Molecular , Conformação Proteica , Prótons , Homologia de Sequência de Aminoácidos , Zinco/química , Zinco/metabolismoRESUMO
Tumor-to-tumor metastasis is a rare event with meningioma as the recipient tumor accounting for 20% of the reported cases. The most common primary cancers showing this phenomenon are lung and breast cancer. Most lung cancers metastasizing to a meningioma are due to lung adenocarcinoma with the literature containing only 3 prior reports of small-cell lung cancer showing this pattern of spread. Herein, we present the case of a 67-year-old-patient with small-cell lung cancer that developed a metastasis to a meningioma.
RESUMO
The development of ion mobility (IM) MS instruments has the capability to provide an added dimension to peptide analysis pipelines in proteomics, but, as yet, there are few software tools available for analysing such data. IM can be used to provide additional separation of parent ions or product ions following fragmentation. In this work, we have created a set of software tools that are capable of converting three dimensional IM data generated from analysis of fragment ions into a variety of formats used in proteomics. We demonstrate that IM can be used to calculate the charge state of a fragment ion, demonstrating the potential to improve peptide identification by excluding non-informative ions from a database search. We also provide preliminary evidence of structural differences between b and y ions for certain peptide sequences but not others. All software tools and data sets are made available in the public domain at http://code.google.com/p/ion-mobility-ms-tools/.
Assuntos
Biologia Computacional/métodos , Bases de Dados de Proteínas , Espectrometria de Massas/métodos , Peptídeos/química , Software , Sequência de Aminoácidos , Humanos , Modelos Lineares , Dados de Sequência Molecular , Peptídeos/análiseAssuntos
Padrões de Prática Médica/normas , Prática Profissional/normas , Cirurgia Bucal/normas , Conhecimentos, Atitudes e Prática em Saúde , Humanos , Cirurgiões Bucomaxilofaciais/psicologia , Avaliação de Resultados em Cuidados de Saúde , Planejamento de Assistência ao Paciente , Fatores de RiscoAssuntos
Avaliação de Resultados em Cuidados de Saúde , Assistência ao Paciente/normas , Cirurgia Bucal/normas , Conhecimentos, Atitudes e Prática em Saúde , Humanos , Cirurgiões Bucomaxilofaciais/psicologia , Planejamento de Assistência ao Paciente , Administração da Prática Odontológica/normas , Fatores de Risco , Resultado do TratamentoRESUMO
Vocalizations of Indo-Pacific humpback dolphins (Sousa chinensis) in west Hong Kong waters were described from 12 recordings in 2010. A broadband hydrophone system recorded sounds. Vocalizations were characterized as broadband click trains, burst pulses, and narrowband frequency modulated sounds, including whistles generally similar to those of some other delphinid cetaceans. A comparison of results to previous humpback dolphin sound descriptions for Moreton Bay, Australia found broad similarities except for the apparent absence of "quacks" and "grunts" in the present study, which are of low frequency and thus were possibly masked by anthropogenic and other low frequency noise in the Hong Kong habitat.
Assuntos
Golfinhos/fisiologia , Vocalização Animal/fisiologia , Animais , Hong Kong , Oceano Índico , Oceano Pacífico , Mascaramento Perceptivo/fisiologia , Espectrografia do Som , Especificidade da EspécieRESUMO
Background: There is evidence that high-fibre diets have significant health benefits, although the effect of increasing fibre on individuals' appetite, satiety, and gastrointestinal comfort is not well established, nor is its acceptability and feasibility. Methods: This mixed-methods feasibility randomised control trial included 38 participants allocated to one of three conditions: FibreMAX (two daily servings of 25 g of BARLEYmax®), FibreGRAD (two daily servings with the amount of fibre gradually increased), and Control (two daily servings totalling 25 g of placebo product). Participants completed a food diary at baseline. The Hunger and Fullness Questionnaire and questions regarding gastrointestinal response were completed at baseline and at the end of each week. Participants completed the acceptability of intervention measure and engaged in a semi-structured interview, following trial completion. Results: The qualitative data suggested that increased fibre influenced appetite and fullness perceptions. Baseline fibre consumption and the method of increased fibre increase did not influence our findings. The qualitative results also indicated that the fibre intake was perceived as beneficial to well-being; it influenced feelings of hunger and caused some minor acute gastrointestinal symptoms that dissipated after a short adaption period. Conclusion: This study suggests that increasing fibre intake through BARLEYmax® is a safe intervention that is acceptable to participants.
Assuntos
Apetite , Saciação , Estudos Cross-Over , Fibras na Dieta , Ingestão de Energia , Estudos de Viabilidade , Humanos , FomeRESUMO
In this paper, we discuss the challenge of large-scale quantification of a proteome, referring to our programme that aims to define the absolute quantity, in copies per cell, of at least 4000 proteins in the yeast Saccharomyces cerevisiae. We have based our strategy on the well-established method of stable isotope dilution, generating isotopically labelled peptides using QconCAT technology, in which artificial genes, encoding concatenations of tryptic fragments as surrogate quantification standards, are designed, synthesised de novo and expressed in bacteria using stable isotopically enriched media. A known quantity of QconCAT is then co-digested with analyte proteins and the heavy:light isotopologues are analysed by mass spectrometry to yield absolute quantification. This workflow brings issues of optimal selection of quantotypic peptides, their assembly into QconCATs, expression, purification and deployment.
Assuntos
Marcação por Isótopo/métodos , Proteômica/métodos , Proteínas de Saccharomyces cerevisiae/análise , Biologia de Sistemas/métodos , Escherichia coli/metabolismo , Espectrometria de Massas , Fragmentos de Peptídeos/análiseRESUMO
Epithelial cell junctions are essential for cell polarity, adhesion and morphogenesis. We have analysed VAB-9, a cell junction protein in Caenorhabditis elegans. VAB-9 is a predicted four-pass integral membrane protein that has greatest similarity to BCMP1 (brain cell membrane protein 1, a member of the PMP22/EMP/Claudin family of cell junction proteins) and localizes to the adherens junction domain of C. elegans apical junctions. Here, we show that VAB-9 requires HMR-1/cadherin for localization to the cell membrane, and both HMP-1/alpha-catenin and HMP-2/beta-catenin for maintaining its distribution at the cell junction. In vab-9 mutants, morphological defects correlate with disorganization of F-actin at the adherens junction; however, localization of the cadherin-catenin complex and epithelial polarity is normal. These results suggest that VAB-9 regulates interactions between the cytoskeleton and the adherens junction downstream of or parallel to alpha-catenin and/or beta-catenin. Mutations in vab-9 enhance adhesion defects through functional loss of the cell junction genes apical junction molecule 1 (ajm-1) and discs large 1 (dlg-1), suggesting that VAB-9 is involved in cell adhesion. Thus, VAB-9 represents the first characterized tetraspan adherens junction protein in C. elegans and defines a new family of such proteins in higher eukaryotes.
Assuntos
Proteínas de Caenorhabditis elegans/isolamento & purificação , Caenorhabditis elegans/metabolismo , Adesão Celular/genética , Epiderme/metabolismo , Células Epiteliais/metabolismo , Junções Intercelulares/metabolismo , Proteínas de Membrana/isolamento & purificação , Citoesqueleto de Actina/metabolismo , Junções Aderentes/genética , Junções Aderentes/metabolismo , Animais , Caderinas/metabolismo , Caenorhabditis elegans/genética , Caenorhabditis elegans/ultraestrutura , Proteínas de Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/metabolismo , Tamanho Celular/genética , Claudina-1 , Proteínas do Citoesqueleto/metabolismo , DNA Complementar/análise , DNA Complementar/genética , Epiderme/ultraestrutura , Células Epiteliais/ultraestrutura , Junções Intercelulares/genética , Junções Intercelulares/ultraestrutura , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Microscopia Eletrônica , Dados de Sequência Molecular , Mutação/genética , Homologia de Sequência de Aminoácidos , Homologia de Sequência do Ácido Nucleico , Transativadores/metabolismo , alfa Catenina , beta CateninaRESUMO
Selected reaction monitoring mass spectrometry has been combined with the use of an isotopically labelled synthetic protein, made up of proteotypic tryptic peptides selected from parasite proteins of interest. This allows, for the first time, absolute quantification of proteins from Plasmodium falciparum. This methodology is demonstrated to be of sufficient sensitivity to quantify, even within whole cell extracts, proteins of low abundance from the folate pathway as well as more abundant "housekeeping" proteins.
Assuntos
Espectrometria de Massas/métodos , Parasitologia/métodos , Plasmodium falciparum/química , Proteínas de Protozoários/análiseRESUMO
Individuals often face unpredictable and harsh environments, presenting them with novel ecological problems. Behaviour can provide an adaptive response in such conditions and where these conditions vary between populations, we may predict development and evolution to shape differences in behaviour such as exploration, innovation, and learning, as well as other traits. Here, we compared in the wild the maze swimming performance of groups of female guppies from two Trinidadian populations that differ in numerous ecological characteristics, the Upper and Lower Aripo river. Compared to Upper Aripo fish, Lower Aripo fish were slower to complete the maze, our measure of propensity to innovate, and scored lower on a combined measure of activity and exploration. More active-exploratory groups were faster to complete the maze, but only in the Lower Aripo. We found no evidence for learning the maze. Our results suggest that activity-exploratory and innovative propensities can vary between populations, as can predictors of innovation. These findings are consistent with high predation risk shaping decreased activity-exploratory propensities, but further population comparisons are required to reliably determine the drivers of the observed population difference. Our results emphasize that individual and population differences in activity-exploration and innovation can be shaped by numerous factors.
Assuntos
Poecilia , Animais , Evolução Biológica , Feminino , Humanos , Fenótipo , Comportamento Predatório , NataçãoRESUMO
Cervical cancer is preventable and curable yet causes almost 2000 deaths in Nepali women each year. The present study aims to explore the feasibility and acceptability of a self-sampling-based approach for cervical cancer screening in urban and peri-urban Nepal and develop pathways for self-sampling using a co-design methodology. An iterative design approach was applied. Semi-structured in-depth interviews were conducted with 30 healthy women and four women who had had a prior cancer diagnosis on topics which included: sexual and reproductive health knowledge and human papillomavirus (HPV); use of the internet/social media platforms; their views regarding acceptability and usability of the self-sampling kit and the proposed user journey. Data collection was done between December 2020 and January 2021. Seven medical experts were also interviewed to explore the current service configuration for cervical cancer screening in Nepal. Knowledge regarding HPV and its association with cervical cancer was absent for the majority of participants. Although 70% (n = 21/30) had purchased items online previously, there was a general lack of trust in online shopping. Half of the women (n = 17/30; 56.7%) expressed a willingness to self-sample and provided recommendations to improve the clarity of the instructions. The proposed user journey was considered feasible in the urban area. There is a clear unmet need for information about HPV and alternative cervical screening options in Nepal. An online pathway for self-sampling service delivery to urban women is feasible but will need to be optimally designed to address barriers such as confidence in self-sampling and trust in online purchasing.
Assuntos
Alphapapillomavirus , Neoplasias do Colo do Útero , Detecção Precoce de Câncer , Estudos de Viabilidade , Feminino , Humanos , Nepal , Papillomaviridae , Neoplasias do Colo do Útero/diagnósticoRESUMO
BACKGROUND: Plasmodium species are difficult to study using proteomic technology because they contain large amounts of haemoglobin-derived products (HDP), generated by parasite breakdown of host haemoglobin. HDP are known to interfere with isoelectric focussing, a cornerstone of fractionation strategies for the identification of proteins by mass spectrometry. In addition to the challenge presented by this material, as in most proteomes, there exists in this parasite a considerable dynamic range between proteins of high and low abundance. The enzymes of the folate pathway, a proven and widely used drug target, are included in the latter class. METHODS: This report describes a work-flow utilizing a parasite-specific extraction protocol that minimizes release of HDP into the lysate, followed by in-solution based OFFGEL™ electrophoresis at the protein level, trypsin digestion and mass spectrometric analysis. RESULTS: It is demonstrated that, by removing HDP from parasite lysates, OFFGEL™-mediated protein separation is able to deliver reduced complexity protein fractions. Importantly, proteins with similar and predictable physical properties are sharply focussed within such fractions. CONCLUSIONS: By following this novel workflow, data have been obtained which allow the unequivocal experimental identification by mass spectrometry of four of the six proteins involved in folate biosynthesis and recycling.